Processing of nucleic acids for further studies, including sequencing, hybridization or PCR (polymerase chain reaction), will require isolation and purification of the plasmid DNA from chromosomal DNA, RNA, proteins and other contaminants present in bacterial cells. Most of these manipulations will require highly purified plasmid DNA.
A number of plasmid purification methods have been developed over the years. These methods include organic solvent extraction, separation according to buoyant density, size exclusion chromatography, and ion-exchange chromatography.
Alkaline lysis/organic solvent extraction has been the classic method to obtain purified plasmid DNA. The procedure involves numerous extraction and precipitation steps using organic solvents such as chloroform or phenol. Although an excellent method for obtaining significant quantities of highly purified plasmid DNA, it has serious drawbacks. The procedure is tedious and lengthy. There is often organic solvent contamination in the final product that must be removed before further studies. The procedure also has significant safety concerns due to the use of noxious chemicals in the process.
Separation according to buoyant density is another popular method of plasmid purification and provides high purity DNA. This method involves mixing a crude preparation containing the plasmid DNA with ethidium bromide dye and then over-layering the sample on top of a cesium chloride (CsCl) solution of higher buoyant density. This mixture is centrifuged at high speed to form a gradient of CsCl of increasing buoyant density. Macromolecules in the sample separate according to their buoyant density in the CsCl gradient, and a discreet band of plasmid DNA can be isolated from other contaminants, such as bacterial DNA. RNA is collected on the bottom of the tube as a pellet. However, this procedure is extremely time consuming, involving often 24 to 48 hours of centrifugation for a single sample, in order to form the CsCl gradient. Furthermore, the resulting band of isolated plasmid DNA is contaminated with ethidium bromide and residual CsCl and must be subjected to multiple organic washes prior to use.
Chromatographic procedures are also typical methods for purifying plasmid DNA. These methods include size exclusion chromatography, where separation is based on the size and solution conformation differences between linear and circular DNA, and ion-exchange chromatography, where separation is based on charge density differences between nucleic acids and other contaminating macromolecules.
Ion-exchange protocols have gained widespread acceptance as the method of choice for rapid isolation and purification of plasmid DNA. Using an appropriate buffer system, the anionic plasmid DNA is adsorbed to an ion-exchange support matrix which has been functionalized with anion exchange groups that bear a net positive charge under the buffer conditions used. The bound DNA can then be released from the ion-exchange surface by increasing the concentration of a suitable counter-ion, e.g., chloride (Cl.sup.-). Variations on anionic exchange separations are described, for example, in U.S. Pat. No. 4,997,932 (porous beads with an anion-exchange surface for purifying nucleic acid); U.S. Pat. No. 4,935,342 (anion-exchange column); Warren and Merion, BioChrom. 3:118-126 (1988) (purification of synthetic oligonucleotides by anion-exchange high performance liquid chromatography using DEAE resin columns).